A method for detecting teprenone isomers

Abstract

The present invention belongs to the technical field of chemical detection, and specifically relates to a method for detecting teprenone isomers. Teprenone is a digestive system drug for acid suppression and gastric mucosal protection, and is a mixture of isomers, with the all-trans structure playing the main pharmacodynamic role; therefore, developing a simple and practical method that can effectively separate and detect the two isomers of teprenone, mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E), is of great significance for conducting research on the synthesis of all-trans teprenone and production quality control. The invention discloses a method for detecting teprenone isomers, and high performance liquid chromatography is used to detect two isomers of teprenone, the chromatographic column is a chiral column, CHIRALPAK IB (4.6*250mm, 5μm); the flow rate is 0.7-0.9mL / min; the column temperature is 20-30℃; the injection volume is 10-50μL; the wavelength is 210-220nm; the mobile phase is n-hexane: n-butanol: anhydrous ethanol = (80-75): (15-20): 5; the elution procedure is isocratic elution; the elution times of the two isomers of teprenone, the all-trans and mono-cis, are 8.270min and 9.113min respectively, the separation degree is 2.68, the baseline is stable, and the main peak shape in the test solution is good. Through methodological verification, the method can be applied to the separation and content determination of the two isomers of teprenone, the mono-cis and all-trans, in teprenone bulk drugs or teprenone capsules.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for detecting teprenone isomers. Background Art

[0002] Teprenone is a digestive system drug that acts as an acid suppressant and gastric mucosal protector. Its chemical name is 6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one. Teprenone is actually a mixture of mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) geometric isomers. Its chemical structure information is as follows:

[0003]

[0004] Data show that the main pharmacological effect of the teprenone mixture is the all-trans structure, i.e., Teprenone all-trans (5E, 9E, 13E), CAS 6809-52-5. Therefore, the development of a simple and practical method that can effectively separate and detect the two isomers of teprenone, mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E), is of great significance for the research on the synthesis of all-trans teprenone and the research on production quality control.

[0005] The method for determining the content of teprenone reported in the literature currently determines the total amount of the two isomers. Since the peak times of the two isomers overlap, it is not possible to effectively separate the single cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) geometric isomers and perform effective content determination. The isomer determination method reported in the Japanese Pharmacopoeia uses gas chromatography, which is complicated to operate, time-consuming to detect, and not very operable and popular.

[0006] Therefore, it is necessary to develop a high performance liquid chromatography content determination method that can effectively separate and detect the mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) geometric isomers of teprenone and is more easily operable for the synthesis and quality control of teprenone API isomers. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention discloses a method for detecting teprenone isomers:

[0008] The mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) isomers of teprenone were detected by high performance liquid chromatography. The system suitability solution and the test solution were prepared, and the diluent, system suitability solution and the test solution were injected into the high performance liquid chromatograph respectively, and the content was calculated by the peak area normalization method.

[0009] High performance liquid chromatography conditions: the chromatographic column is a chiral column, CHIRALPAK IB; the flow rate is 0.7-0.9 mL / min; the column temperature is 20-30°C; the mobile phase is n-hexane: n-butanol: anhydrous ethanol = (75-82): (20-13): 5; the elution procedure is isocratic elution;

[0010] The structural formulas of the two isomers of teprenone, mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E), are as follows:

[0011]

[0012] Among them, A is all-trans (5E, 9E, 13E) and B is mono-cis (5Z, 9E, 13E).

[0013] Preferably, the chromatographic column is a chiral column CHIRALPAK IB: 4.6*250 mm, 5 μm.

[0014] Furthermore, the detection wavelength is 210-220 nm; the injection volume is 10-50 μL; and the diluent is n-hexane:n-butanol=(40-60):(60-40).

[0015] Preferably,

[0016] The detection wavelength was 215 nm; the injection volume was 20 μL;

[0017] The diluent is n-hexane: n-butanol = 50:50;

[0018] Flow rate: 0.8 mL / min; column temperature: 25°C.

[0019] Furthermore, the mobile phase is n-hexane: n-butanol: anhydrous ethanol = (82-78): (13-17): 5.

[0020] Preferably, the mobile phase is n-hexane: n-butanol: anhydrous ethanol = 80:15:5;

[0021] Application of any of the above detection methods in the separation and content determination of the two isomers of teprenone, mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) in teprenone bulk drug or teprenone capsules.

[0022] The invention adopts high performance liquid chromatography to separate and determine the content of two isomers of teprenone. The elution times of the all-trans and mono-cis isomers of teprenone are 8.270 min and 9.113 min respectively, the separation degree is 2.68, the baseline is stable, the main peak shape in the test solution is good, and the effective separation and content determination of the all-trans and mono-cis isomers of teprenone within 10 min are achieved, and the detection limit is as low as 2.5 ng / ml. The invention adopts an area normalization method to replace the external standard method of reference substance, which can effectively save the use of expensive isomer reference substances, greatly save the detection cost, and reduce the complicated procedures of isomer reference substance solution preparation and dilution, avoiding the measured value deviation caused by the error generated in the reference substance solution preparation and dilution process. The chromatographic conditions of the invention are simple, and a common ultraviolet detector or a DAD detector can be used. The mobile phase elution program is isocratic elution, and the invention can be applied to a single-pump high performance liquid chromatograph. Through methodological verification, it was shown that this method can be applied to the separation and content determination of mono-cis and all-trans isomers of teprenone in teprenone bulk drug or teprenone capsules. The method is convenient, practical, low-cost and simple to operate, and has important application value for conducting research on the synthesis of all-trans teprenone and production quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the blank solution chromatogram;

[0024] Figure 2 is the system suitability solution chromatogram;

[0025] Figure 3 is the chromatogram of the test solution;

[0026] Figure 4 Chromatogram of the diluted positioning solution for all-trans -teprenone;

[0027] Figure 5 Chromatogram of the solution for the dilution positioning of mono-cis-teprenone;

[0028] Figure 6 The detection limit solution chromatogram. DETAILED DESCRIPTION

[0029] Teprenone is a compound with unclear acidity and alkalinity, and the method of improving the separation effect by using a pH regulator is not obvious. The Japanese Pharmacopoeia uses gas chromatography to separate and detect mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) teprenone. The gas chromatography operation process is complicated, and the GC method takes 45 minutes, which is time-consuming and not widely applicable.

[0030] The invention discloses a method for detecting teprenone isomers. The method comprises the following steps: adopting a high performance liquid chromatography method to detect two isomers of teprenone, preparing a system suitability solution and a test solution; injecting a blank solution, a system suitability solution and a test solution into a high performance liquid chromatograph respectively, and calculating the content by adopting a peak area normalization method, wherein the blank solution is a diluent.

[0031] High performance liquid chromatography conditions: the chromatographic column is a chiral column, Daicel CHIRALPAK IB (4.6*250mm, 5μm); the flow rate is 0.7-0.9mL / min; the column temperature is 20-30°C; the injection volume is 10-50μL; the wavelength is 210-220nm; the mobile phase is n-hexane: n-butanol: anhydrous ethanol = (82-75): (13-20): 5; the elution procedure is isocratic elution; the elution time of mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) teprenone is within 10min; the diluent is n-hexane: n-butanol = (40-60): (60-40).

[0032] The structural formulas of the two isomers of teprenone are as follows:

[0033]

[0034] Note: A is all-trans (5E, 9E, 13E) and B is mono-cis (5Z, 9E, 13E) teprenone.

[0035] Preferably, flow rate: 0.8 mL / min; column temperature: 25°C; injection volume: 20 μL; wavelength: 215 nm; mobile phase is n-hexane: n-butanol: anhydrous ethanol = 80:15:5; the elution times of the all-trans and mono-cis isomers of teprenone are 8.270 min and 9.113 min, respectively.

[0036] The study found that a diluent of n-hexane: n-butanol = (40-60): (60-40) does not affect the effective separation and detection of the two isomers under the above chromatographic conditions; preferably, the diluent is n-hexane: n-butanol = 50:50.

[0037] Example 1-4, Comparative Example 1-2: Screening of Chromatographic Column and Mobile Phase

[0038]

[0039] The experimental test results show that the use of CHIRALPAK IB 4.6×250mm 5μm, flow rate: 0.8mL / min; column temperature: 25℃; injection volume: 20μL; wavelength: 215nm; mobile phase is n-hexane: n-butanol: anhydrous ethanol = 80:15:5; elution time: 15min. The elution times of the two isomers of teprenone, all-trans and mono-cis, are 8.270min and 9.113min respectively, with a separation of 2.68, a stable baseline, and a good peak shape of the main peak in the test solution, which meets the requirements.

[0040] Example 5: Specificity test

[0041] 1.1 Instruments and reagents: Thermo Fisher Scientific 3000 high performance liquid chromatograph (UV detector), Mettler electronic analytical balance (1 / 100,000), ultrasonic cleaner; reference substances of the two isomers of teprenone, all-trans and mono-cis, with a purity of not less than 99%, meeting the quantitative requirements; teprenone API and teprenone capsules provided by a certain manufacturer; other reagents include chromatographic alcohol, analytical alcohol, etc.

[0042] 1.2 Chromatographic conditions: CHIRALPAK IB 4.6×250mm 5μm, mobile phase: n-hexane: n-butanol: anhydrous ethanol = 80:15:5; elution program: isocratic elution; flow rate: 0.8mL / min; column temperature: 25℃; injection volume: 20μL; wavelength: 215nm; the theoretical plate number calculated based on all-trans-teprenone should not be less than 5000.

[0043] 1.3 Preparation of dilution solution: prepare n-hexane: n-butanol at a ratio of 50:50.

[0044] 1.4 Preparation of test solution: Take the Teprenone API, accurately weigh about 50 mg of fine powder, place it in a 50 mL volumetric flask, add diluent to dissolve and make up to the scale, shake well, filter, and use the filtrate as the test solution.

[0045] 1.5 Reference solution of series concentration: Take appropriate amount of all-trans and mono-cis teprenone reference substances respectively, and use diluent to prepare reference stock solution of about 0.5mg / ml. Use reference stock solution to dilute to series reference solution, which can be used as reference positioning solution. Further dilute to prepare detection limit solution.

[0046] 1.6 Preparation of system suitability solution: Take 100ul of each of the all-trans and mono-cis teprenone reference substance stock solutions under 1.5 and dilute them with diluent to a concentration of about 0.5ug / ml.

[0047] 1.7 Specificity test: Take 20ul of diluent, system suitability solution, reference solution, and test solution for analysis. Figure 1-5 As shown, according to the peak elution time of all-trans and mono-cis teprenone in the reference positioning solution, the peak elution time of all-trans and mono-cis teprenone in the system adaptability solution was 8.270min and 9.113min, respectively. The diluent did not interfere with the detection of all-trans and mono-cis teprenone. There was no situation in the test solution that affected the target peaks of all-trans and mono-cis teprenone, and there was no situation where the separation degree of adjacent peaks was less than 1.5, indicating that the specificity of this method was good.

[0048] Example 6: Methodological Validation

[0049] Linear relationship investigation, precision, repeatability, stability, accuracy (recovery rate), durability, etc. are conventional research methods known to analysts in this field. Specifically, relevant experiments are carried out with reference to the 9101 Guidelines for Validation of Analytical Methods in Part IV of the 2020 Edition of the Chinese Pharmacopoeia.

[0050] 1.8 Linear Relationship Investigation

[0051] The calibration curve was drawn with the concentration of the reference substance (ug / mL or ng / ml) as the horizontal coordinate x and the peak area of ​​the reference substance as the vertical coordinate y. The regression equation, correlation coefficient and linear range are shown in Table 1. As can be seen from the table, the linear relationship between all-trans and mono-cis teprenone is good within the corresponding concentration range. The detection limit of all-trans teprenone is 2.5 ng / ml (see Figure 6 ).

[0052] Table 1. Results of the linear relationship study of all-trans and mono-cis teprenone

[0053]

[0054] 1.9 Precision test

[0055] The same test solution was taken and injected 6 times, 20 μL each time. The RSDs of the peak areas of all-trans and mono-cis teprenone were 0.12% and 0.46%, respectively, which met the requirements of the precision test and showed that the instrument had good precision.

[0056] 2.0 Repeatability Test

[0057] Six test solutions were prepared repeatedly according to the method under "1.4", and the measurements were performed separately. The peak areas of the chromatographic peaks were recorded, and the contents of all-trans and mono-cis teprenone were calculated. The RSDs were 0.75% and 0.62%, respectively, indicating that the method of the present invention had good repeatability.

[0058] 2.1 Stability test

[0059] The same test solution was taken and measured at 0h, 2h, 4h, 6h, 10h and 12h, and the RSDs of the peak areas were 0.80% and 0.78%, respectively, indicating that all-trans and mono-cis-teprenone in the sample were stable within 12 hours.

[0060] 2.2 Sample recovery test

[0061] Take an appropriate amount of teprenone raw material with known content, weigh an appropriate amount respectively, accurately add all-trans and mono-cis teprenone reference solution, prepare 6 portions of 100% solution or 9 portions of 80%, 100%, and 120% solution according to the method under "1.4", and measure according to the chromatographic conditions under "1.2". After calculation, the sample recovery rates of all-trans and mono-cis teprenone were 99.32% and 98.91%, respectively, and the RSDs were 1.08% (n=6) and 1.44% (n=6), respectively, and the sample recovery rates were good.

[0062] 2.3 Durability test

[0063] A durability study was conducted with column temperatures of 20°C and 30°C, flow rates of 0.7mL / min and 0.9mL / min, injection volumes of 10μL and 50μL, detection wavelengths of 210nm and 220nm, and a mobile phase ratio of n-hexane: n-butanol: anhydrous ethanol = (80±2): (15±2): 5.

[0064] The results showed that the flow rate change (0.7mL / min~0.9mL / min) and column temperature change (20℃~30℃) affected the elution time of all-trans and mono-cis teprenone, but the elution time of mono-cis and all-trans teprenone was within 12min; the flow rate change (0.7mL / min~0.9mL / min) and column temperature change (20℃~30℃) also affected the separation degree, but did not affect the effective separation of mono-cis and all-trans teprenone (the separation degree was greater than 1.5); the detection wavelength (210nm~220nm) had no significant effect on mono-cis and all-trans teprenone. Under the above conditions, the chromatogram baseline was good and the separation effect was good.

[0065] The results of the study on the fine adjustment of the mobile phase ratio in combination with Examples 1-4 and the durability test show that when the mobile phase is in the range of n-hexane: n-butanol: anhydrous ethanol = (75-82): (20-13): 5, the separation degree of mono-cis and all-trans teprenone is greater than 1.5, and the separation and detection of mono-cis and all-trans teprenone can be achieved; preferably, when the mobile phase ratio is n-hexane: n-butanol: anhydrous ethanol = (80±2): (15±2): 5, that is, the mobile phase ratio is n-hexane: n-butanol: When anhydrous ethanol = (82-78): (13-17): 5, the separation degrees of mono-cis and all-trans teprenone are both greater than 2.0, and the baseline separation effect of the two isomers is better; more preferably, when the mobile phase ratio is n-hexane: n-butanol: anhydrous ethanol = 80:15:5, as shown in Example 1, the elution times of the all-trans and mono-cis isomers of teprenone are 8.270 min and 9.113 min, respectively, with a separation degree of 2.68, a stable baseline, and a good peak shape of the main peak in the test solution.

[0066] The above research results show that the detection method of the present invention has good durability.

[0067] 2.4 Determination of Teprenone API Samples

[0068] Three batches of teprenone API were accurately weighed, and the test solution was prepared according to the method under "1.4". The chromatographic conditions under "1.2" were used for determination. The separation degree of all-trans and mono-cis teprenone and the adjacent impurity peaks was greater than 1.5. The contents of all-trans and mono-cis teprenone in the samples were calculated by external standard method and area normalization method, respectively. The results are shown in Table 2.

[0069] Table 2. Determination results of all-trans and mono-cis teprenone in teprenone API

[0070]

[0071] The data results show that there is no significant difference between the determination results using the area normalization method and the reference external standard method (the RD of all-trans teprenone is less than 2%, and the RD of mono-cis teprenone is less than 11%, both of which meet the requirements of General Chapter 9101 of Part IV of the Chinese Pharmacopoeia), indicating that the detection method of the present invention using the area normalization method to calculate the content realizes the effective separation and content determination of the two isomers of all-trans and mono-cis teprenone in the teprenone raw material, effectively saves the use of expensive isomer reference substances, greatly saves the detection cost, and also reduces the complicated process of preparing and diluting the isomer reference solution, avoiding the deviation of the measured value caused by the error generated in the preparation and dilution process of the reference solution.

[0072] The above test results also show that the teprenone raw material developed by this manufacturer has achieved the synthesis and production of all-trans teprenone.

[0073] 2.5 Determination of Teprenone Capsules Samples

[0074] Furthermore, the test solution of the teprenone capsule sample was prepared according to the method under item "1.4", and the test was carried out according to the chromatographic conditions under item "1.2". The separation degrees of all-trans and mono-cis teprenone and the adjacent impurity peaks were both greater than 1.5, indicating that the method of calculating the content by area normalization method in the present invention can also be applied to the separation and content determination of all-trans and mono-cis teprenone in teprenone capsules.

[0075] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, other relevant modifications can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for detecting teprenone isomers, characterized in that: The mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) isomers of teprenone were detected by high performance liquid chromatography. The system suitability solution and the test solution were prepared, and the diluent, system suitability solution and the test solution were injected into the high performance liquid chromatograph respectively, and the content was calculated by the peak area normalization method. HPLC conditions: chromatographic column is chiral column CHIRALPAK IB; flow rate is 0.7-0.9 mL / min; column temperature is 20-30°C; mobile phase is n-hexane: n-butanol: anhydrous ethanol = (75-82): (20-13): 5; elution procedure is isocratic elution; The structural formulas of the two isomers of teprenone, mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E), are as follows: ； Among them, A is all-trans (5E, 9E, 13E) and B is mono-cis (5Z, 9E, 13E).

2. The method for detecting a teprenone isomer according to claim 1, characterized in that: The chromatographic column is a chiral column, CHIRALPAK IB: 4.6*250mm, 5μm.

3. The method for detecting a teprenone isomer according to claim 1, characterized in that: The detection wavelength is 210-220 nm; the injection volume is 10-50 μL; the diluent is n-hexane: n-butanol=(40-60):(60-40).

4. The method for detecting a teprenone isomer according to claim 3, characterized in that: The detection wavelength is 215 nm.

5. The method for detecting a teprenone isomer according to claim 3, characterized in that: The injection volume was 20 μL; the diluent was n-hexane: n-butanol = 50:

50.

6. The method for detecting a teprenone isomer according to claim 1, characterized in that: Flow rate: 0.8 mL / min; column temperature: 25°C.

7. The method for detecting a teprenone isomer according to claim 1, characterized in that: The mobile phase is n-hexane: n-butanol: anhydrous ethanol = (82-78): (13-17):

5.

8. The method for detecting teprenone isomers according to claim 7, characterized in that: The mobile phase was n-hexane: n-butanol: anhydrous ethanol = 80:15:

5.

9. Use of the detection method according to any one of claims 1 to 8 in the separation and content determination of the two isomers of teprenone, mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) in teprenone bulk drug.

10. Use of the detection method according to any one of claims 1 to 8 in the separation and content determination of the mono-cis (5Z, 9E, 13E) and all-trans (5E, 9E, 13E) isomers of teprenone in teprenone capsules.

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